Utility of Liquid Biopsy by Improved PNA-LNA PCR Clamp Method for Detecting EGFR Mutation at Initial Diagnosis of Non–Small-Cell Lung Cancer: Observational Study of 190 Consecutive Cases in Clinical Practice

Background

The clinical benefit of liquid biopsy for unselected patients at initial diagnosis has thus far been unclear. We aimed to evaluate the utility of liquid biopsy at initial diagnosis, as well as the efficacy of epidermal growth factor receptor–tyrosine kinase inhibitor (EGFR-TKI) based on liquid biopsy results in clinical practice, using the improved peptide nucleic acid–locked nucleic acid (PNA-LNA) PCR clamp method.

Differential effects of organotin compounds on voltage-gated potassium currents in lymphocytes and neuroblastoma cells

Effects of organotin compounds were studied on voltage-gated K⁺ current in whole-cell voltage clamped lymphocytes and in NlE-115 neuroblastoma cells. In human peripheral blood lymphocytes the immunotoxic compounds dibutyltinchloride (DBT, 2.5 M) and triphenyltinchloride (TPhT, 2.5 M) decrease the peak amplitude of the K⁺ current and prolong time to peak. Tributyltinchloride (TBT, 2.5 M) decreases the K⁺ current to a greater extent than DBT and TPhT, without affecting the time to peak. The neurotoxic organotin compound trimethyltinchloride (TMT, 2.5 M) does not affect the voltage-gated K⁺ current in lymphocytes. Similar effects of DBT were observed in freshly isolated and PHA-activated human lymphocytes and with rat thymocytes. On the other hand, in mouse NIE-115 neuroblastoma cells, none of the organotin compounds altered the voltage-dependent K⁺ current.In human lymphocytes DBT affects both the peak amplitude and the time to peak of the K⁺ current in a concentration-dependent manner. At the maximum concentration of 10 M tested, the peak amplitude of the K⁺ current was reduced to 22 ± 4% of the control current. The IC₅₀ and slope factor for block of the peak outward current by DBT amounts to 6.7 ± 0.4 M, and 2.7 ± 0.4, respectively. The delay in K⁺ current activation does not saturate. At 10 M DMT increases the time to peak to 332 ± 12% of the control value. The present results suggest that the effects by DBT originate from two separate interactions with the voltage-gated K⁺ channel at the extracellular site of the membrane: a direct effect on the closed K⁺ channel causing a delay in current activation and a membrane-related effect causing inhibition of the K⁺ current. The differential effects of the organotin compounds may relate to their differential toxicological action.     

Effects of polyamines on voltage-activated calcium channels in guinea-pig intestinal smooth muscle

Effects of polyamines on the spontaneous mechanical and electrical activity of guinea-pig intestinal smooth muscle were studied. Spermine and spermidine inhibited action potential generation and contractions, while putrescine had no effect. Single smooth muscle cells were isolated from the longitudinal muscle layer of the guinea-pig ileum. Whole-cell voltage-clamp experiments were carried out to investigate the effects of polyamines on current through voltage-activated Ca²⁺ channels. Spermine and spermidine (0.1–1 mM) reduced the inward current in a concentration-dependent manner. Spermine blocked current activated by the dihydropyridine agonist BAY K 8644 (1 M), whereas no additional inhibition by spermine was seen after blockage of dihydropyridine-sensitive channels by nifedipine (0.1 M). Inhibition by spermine or spermidine did not shift the peak of the current voltage relation of the inward current. Steady-state activation and inactivation relationships were not affected and thus the amplitude, but not the voltage dependence, of the window current responsible for Ca²⁺ inflow during sustained depolarization was affected. Putrescine (1 mM) had no significant effect on the inward current. These results suggest that spermine and spermidine inhibit contraction in spontaneously active intestinal smooth muscle by inhibiting Ca²⁺ current responsible for generation of action potentials.     

Orphenadrine is an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist: binding and patch clamp studies

Summary Orphenadrine has been used as an antiparkinsonian, antispastic and analgesic drug for many years. Here we show that orphenadrine inhibits [³H]MK-801 binding to the phencyclidine (PCP) binding site of the N-methyl-D-aspartate (NMDA)-receptor in homogenates of postmortem human frontal cortex with a K_i-value of 6.0 ± 0.7 M. The NMDA receptor antagonistic effects of orphenadrine were assessed using concentration- and patch-clamp techniques on cultured superior colliculus neurones. Orphenadrine blocked open NMDA receptor channels with fast kinetics and in a strongly voltage-dependent manner. The IC₅₀-value against steady state currents at –70mV was 16.2 ± 1.6 M (n=6). Orphenadrine exhibited relatively fast, concentration-dependent open channel blocking kinetics (K_on 0.013 ± 0.002 10⁶M^–1S^–1) whereas the offset rate was concentration-independent (K_off 0.230 ± 0.004 S^–1). Calculation of the ratio K_off/K_on revealed an apparent K_d-value of 17.2 M which is nearly identical to the IC₅₀ calculated at equilibrium.     

Involvement of voltage-dependent potassium channels in the EDHF-mediated relaxation of rat hepatic artery

1. In the rat hepatic artery, the acetylcholine-induced relaxation mediated by endothelium-derived hyperpolarizing factor (EDHF) is abolished by a combination of apamin and charybdotoxin, inhibitors of small (SK_Ca) and large (BK_Ca) conductance calcium-sensitive potassium (K)-channels, respectively, but not by each toxin alone. The selective BK_Ca inhibitor iberiotoxin cannot replace charybdotoxin in this combination. Since delayed rectifier K-channels (K_V) represent another target for charybdotoxin, we explored the possible involvement of K_V in EDHF-mediated relaxation in this artery.
2. The K_V inhibitors, agitoxin-2 (0.3 μM), kaliotoxin (0.3 μM), β-dendrotoxin (0.3 μM), dofetilide (1 μM) and terikalant (10 μM), each in combination with apamin (0.3 μM) had no effect on the EDHF-mediated relaxation induced by acetylcholine in the presence of N^ω-nitro-L-arginine (0.3 mM) and indomethacin (10 μM), inhibitors of nitric oxide (NO) synthase and cyclo-oxygenase, respectively (n=2–3). Although the K_V inhibitor margatoxin (0.3 μM) was also without effect (n=5), the combination of margatoxin and apamin produced a small inhibition of the response (pEC₅₀ and E_max values were 7.5±0.0 and 95±1% in the absence and 7.0±0.1 and 81±6% in the presence of margatoxin plus apamin, respectively; n=6; P<0.05).
3. Ciclazindol (10 μM) partially inhibited the EDHF-mediated relaxation by shifting the acetylcholine-concentration-response curve 12 fold to the right (n=6; P<0.05) and abolished the response when combined with apamin (0.3 μM; n=6). This combination did not inhibit acetylcholine-induced relaxations mediated by endothelium-derived NO (n=5).
4. A 4-aminopyridine-sensitive delayed rectifier current (I_K(V)) was identified in freshly-isolated single smooth muscle cells from rat hepatic artery. None of the cells displayed a rapidly-activating and -inactivating A-type current. Neither charybdotoxin (0.3 μM; n=3) nor ciclazindol (10 μM; n=5), alone or in combination with apamin (0.3 μM; n=4–5), had an effect on I_K(V). A tenfold higher concentration of ciclazindol (0.1 mM, n=4) markedly inhibited I_K(V), but this effect was not increased in the additional presence of apamin (0.3 μM; n=2).
5. By use of membranes prepared from rat brain cortex, [¹²⁵I]-charybdotoxin binding was consistent with an interaction at a single site with a K_D of approximately 25 pM. [¹²⁵I]-charybdotoxin binding was unaffected by iberiotoxin (0.1 μM, n=6), but was increased by apamin in a concentration-dependent manner (E_max 43±10%, P<0.05 and pEC₅₀ 7.1±0.2; n=7–8). Agitoxin-2 (10 nM) displaced [¹²⁵I]-charybdotoxin binding by 91±3% (n=6) and prevented the effect of apamin (1 μM; n=6).
6. It is concluded that the EDHF-mediated relaxation in the rat hepatic artery is not mediated by the opening of either K_V or BK_Ca. Instead, the target K-channels for EDHF seem to be structurally related to both K_V and BK_Ca. The possibility that a subtype of SK_Ca may be the target for EDHF is discussed.

     

Mg~(2+)对猪冠脉平滑肌细胞钙激活钾通道的激活作用的初步研究

目的：研究Mg2+对猪冠脉平滑肌细胞钙激活钾通道的作用,以揭示Mg2+扩血管作用的电生理机制。方法：应用膜片钳制技术inside－out方式记录冠脉平滑肌上的单通道活动,经PCLAMP软件进行微机采样储存数据和数据的分析处理。结果：Mg2+对猪冠脉平滑肌钙激活钾通道具有明显激活作用：增强通道开放概率P0,增加膜片通道开放数目。结论：Mg2+对钙激活钾通道的激活作用可能为Mg2+调节心血管活动的重要机制之一。     

钙激活钾通道在钙离子致神经细胞损伤中的作用

采用膜片钳单通道记录方法,观察原代培养新生ＳＤ大鼠皮层神经元的钙激活钾通道（ＫＣａ）特征及胞内不同游离钙水平对通道开放动力学的调节。结果显示：培养ＳＤ大鼠皮层神经元的ＫＣａ以大电导活动占优势,胞内游离钙为１０－８ｍｏｌ／Ｌ时,通道几乎不开放,在１０－６ｍｏｌ／Ｌ时达最大激活。由此表明神经元上ＫＣａ通道开放概率明显依赖于胞浆中钙离子和膜电位,ＫＣａ对胞内钙离子浓度变化极为敏感。钾通道的开放剂可能有一定的神经细胞保护作用。     

银川市城区大气PM_(2.5)中金属元素污染特征及来源分析

目的探讨银川市大气PM_(2.5)中金属元素来源及各来源所占比例,为金属元素污染控制提供科学依据。方法在银川市城区设置2个监测点,2015年每月定期采集大气PM_(2.5)样品,共采集样品164份,分析锑(Sb)、铝(Al)、砷(As)、镉(Cd)、铬(Cr)、汞(Hg)、铅(Pb)、锰(Mn)、镍(Ni)、硒(Se)、铊(Tl)11种金属元素含量及来源。结果 PM_(2.5)质量浓度及部分金属元素含量具有明显的季节变化特征,夏秋低、冬春高。PM_(2.5)中金属元素平均质量浓度顺序:AlPbMnAsTbCdCrSeTlNiHg,环境空气中Pb年均浓度和季平均浓度均未超标。Pb、Tl、Cd、Mn、Se、As主要来源为人为源,主要受到交通源、工业源及燃烧源等人为污染影响。结论银川市大气PM_(2.5)重金属元素的污染特征和来源有其自身的地域性特征。PM_(2.5)质量浓度及金属元素含量受供暖期影响,主要来源于人为污染。     

Use dependence of sodium current inhibition by tetrodotoxin in rat cardiac muscle: influence of channel state

Tetrodoxin (TTX) is known to cause a voltage-and frequency-dependent inhibition of the rapid inward sodium current (I _Na) of cardiac muscle. This effect was studied by means of the loose-patch-clamp method on intact rat papillary muscle. The availability curve of the fast sodium system, determined by variation of the holding potential, is shifted in the presence of TTX (5.5 mol · l^–1) by 17 mV to more negative potentials. With clamp pulses of 5 ms duration to 0 mV, a frequency-dependent reduction of I _Na by TTX is found above 0.1 Hz that saturates at about 10 Hz. This frequency-dependent block was further analysed using trains of pulses (10 Hz) of various durations (minimum 50 s), which allow TTX to equilibrate with channel states reached early during activation. The results show that more than 90% of the frequency-dependent block is attained with pulses of 1 ms duration. An analysis according to the guarded receptor hypothesis reveals that these results are well described by TTX binding to inactivated, activated and probably preactivated channel states.